Methods and compositions for modifying assembly-activating protein (aap)-dependence of viruses

ABSTRACT

Methods and compositions are provided that can be used to modify the assembly activating protein (AAP)-dependence of an adeno-associated virus (AAV).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/409,317, filed May 10, 2019, which is a continuation of InternationalPatent Application No. PCT/US2018/032166, filed on May 10, 2018, whichclaims the benefit of priority under 35 U.S.C. § 119(e) of U.S.Application No. 62/669,901, filed on May 10, 2018, and U.S. ApplicationNo. 62/504,318, filed on May 10, 2017, all three of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 11, 2021, isnamed Sequence_Listing.txt and is 19,481 bytes in size.

TECHNICAL FIELD

This disclosure generally relates to viral vector systems.

BACKGROUND

Adeno-associated virus (AAV) is a leading platform in therapeutic genetransfer, primarily for in vivo gene therapy approaches. Whilepreclinical and clinical studies continue to demonstrate AAV's potentialas a reagent for safe and efficient gene delivery to alleviate a numberof diseases, a bottleneck to its broader application is the productionof sufficient vector quantities to treat these patient populations.

SUMMARY

In general, this disclosure describes and demonstrates the utility of aparticular sequence motif within an AAV capsid protein that enables theassembly-activating protein (AAP)-dependence of the AAV to be modified.Thus, this sequence motif can be used to address and alleviate at leastone of the bottlenecks encountered in the production of virus vectors.In particular, this disclosure describes a minimal motif defined througha novel phenotype-to-phylogeny mapping method that can be used to modifythe AAP-dependence of a virus. Briefly, a number of ancestral AAVs thathave been developed (see, for example, WO 2015/054653 and WO2017/019994, which are incorporated herein by reference in theirentirety) were used to examine AAP dependence across a wide structuraldifferential. This analysis allowed for the identification of a minimalmotif that determines AAP dependency.

In one aspect, the disclosure features adeno-associated virus (AAV)capsid polypeptides including an amino acid sequence having at least 95%sequence identity (e.g., at least 99% sequence identity) to the aminoacid sequence of SEQ ID NO: 3. In some embodiments, the AAV capsidpolypeptide has the amino acid sequence of SEQ ID NO:3. In someembodiments, the AAV capsid polypeptides are encoded by the nucleic acidsequence of SEQ ID NO: 4. In some embodiments, the AAV capsidpolypeptide has the amino acid sequence of SEQ ID NO:1, but contains theamino acid residues at the indicated positions shown in Table 1 for“independence” or “dependence” with respect to AAP.

TABLE 1 Motif for Modifying AAP-Dependence Residue identity and positionin Anc80, Anc81, Aligns to this residue in Anc82, Anc84, rh10 andAnc110, rh8, and AAV9 Site AAV8 “Independence” “Dependence”  1 K163 T162(S in AAV9)  2 A206 S205  3 K478 R476  4 L481 V479 (Tin AAV9)  5 V520 (Ain AAV8) M518  6 T528 S526  7 L586 N584 (H in AAV9)  8 A592-P593Q590-A591  9 N599-S600 H597-N598 (QN in AAV9) 10 A603 V601 (I in AAV9)

This disclosure also features virus particles including any of theadeno-associated virus (AAV) capsid polypeptides described herein. Suchvirus particles can further include a transgene.

In another aspect, the disclosure features nucleic acid moleculesincluding a nucleic acid sequence having at least 95% sequence identity(e.g., at least 99% sequence identity) to the nucleic acid sequence ofSEQ ID NO: 4 and encoding an adeno-associated virus (AAV) capsidpolypeptide. In some embodiments, the nucleic acid molecule has thenucleic acid sequence of SEQ ID NO:4. In some embodiments, the nucleicacid molecule encodes the amino acid sequence of SEQ ID NO: 3.

The disclosure also provides vectors including any of the nucleic acidmolecules described herein, as well as host cells including any of thenucleic acid molecules and/or vectors described herein. In someembodiments, the host cell is a packaging cell.

In another aspect, the disclosure features packaging cells including anucleic acid molecule encoding an adeno-associated virus (AAV) capsidpolypeptide, wherein the AAV capsid polypeptide has at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 3. In someembodiments, the packaging cell lacks the assembly activating protein(AAP).

In another aspect, the disclosure includes methods of reducing theassembly activating protein (AAP)-dependence of an adeno-associatedvirus (AAV). Such methods include providing an AAV having a capsidpolypeptide that has at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 3.

In yet another aspect, the disclosure features methods of relieving, atleast partially, the assembly activating protein (AAP)-dependence of anadeno-associated virus (AAV), the method including: incorporating acapsid polypeptide into the AAV that has at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 3.

In still another aspect, the disclosure provides methods of engineeringan adeno-associated virus (AAV) to reduce its dependence on assemblyactivating protein (AAP), including: engineering an AAV that comprises acapsid polypeptide that has at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 3.

Any of the methods described herein can further include culturing theadeno-associated virus (AAV) in the absence of the assembly activatingprotein (AAP). Any of the methods described herein further can includesequencing the engineered adeno-associated virus (AAV). Any of themethods described herein further can include comparing the assemblyactivating protein (AAP)-dependence of the engineered adeno-associatedvirus (AAV) relative to a non-engineered or wild type AAV. Any of themethods described herein further can include aligning the engineeredadeno-associated virus (AAV) with the non-engineered or wild type AAV.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-D are a series of schematic diagrams, gels, and bar graphs ofdata demonstrating that the requirement for AAP ranges broadly acrossall clades of AAV. Panel A are schematics of AAPstop60 and AAP-HAconstruct maps. Black arrows: transcription start sites at p5, p19, andp40 viral promoters. Grey arrows: cap gene product translation startcodons. Early stop codon (red) introduced by site directed mutagenesis˜60 aa into the AAP ORF. HA tag (orange) inserted at a conserved BsiWIsite near AAP C-terminus. Panel B is a photograph of a gel demonstratingthat AAV1 and AAV3 AAP-HA constructs were generated in both WT AAP andAAPstop60 context. Lysates from transfected HEK293 cells were harvestedafter 36 h, clarified by centrifugation, and interrogated for AAP byWestern Blot with anti-HA antibody. Panel C is a graph demonstrating thevector produced from WT or AAPstop60 rep-cap constructs was titrated byqPCR to quantify DNase resistant particles. AAPstop60 titers arereported as a percentage of each WT serotype titer and represent theaverage of at least 3 independent experiments ±SEM. Bar colorcorresponds to heatmap color on y-axis. † AAPstop60 titer belowbackground level for at least one trial (no cap gene control). See Table2 for statistics. Panel D is a graph of vector produced from WT andAAPstop60 constructs of AAV2 and AAV3 were titrated by A20 capsid ELISA,reported as a percentage of WT titer (average of two experiments; seealso FIG. 11).

FIGS. 2A-E are a series of gels and graphs of experimental data showingVP protein levels in natural serotypes. HEK 293 cells were transfectedwith helper and rep-cap plasmids as denoted above the lane: (wt) WT AAP;(s) AAPstop60; (r) AAPstop60 plus a CMV-driven AAP2. Whole cell lysateswere harvested after 36 hours, clarified by centrifugation, and VPlevels interrogated with B1 antibody (VP1/2/3). Actin was used as aloading control. Serotypes of rep-cap plasmids indicated above eachblot, with (Panel A) AAPstop60 titers ≥10% WT titer and (Panel B)AAPstop60 titers <10% WT titer. Panel C is a blot showing RNA quantifiedfrom AAV2 transfections as above, normalized to GAPDH, reported relativeto AAV2 WT. Minor and major splice isoforms, as well as unsplicedtranscript levels, were examined as denoted on x-axis and diagrammed atright; primers indicated by arrows. Graph represents the mean of threeindependent experiments ±SEM; there is no statistically significantdifference between groups (see Table 2 for statistics). Panel D areblots of HEK 293 cells transfected with helper and AAV8 WT or AAPstop60rep-cap plasmids as indicated. At 24 h, AAPstop60 transfected cells weretreated with concentrations of Bortezomib, MLN7243, or Bafilomycin asindicated above lanes, and incubated an additional 8 h before whole celllysates were harvested as in (Panel A) and (Panel B). VP levels wereinterrogated by Western Blot with B1 antibody (top). Blot was strippedand reprobed for Ubiquitin (bottom). Actin was used as a loading control(middle). Panel E are dot blots using lysates from Panel D treated withDMSO or 1 μM Bortezomib, MLN7243, or Bafilomycin as listed below wereassayed for the presence of assembled capsids by dot blot with the ADK8antibody (recognizes a conformational epitope only present in assembledAAV8 capsids). The experiment was repeated in the presence of AAP tocontrol for any effects of the drugs on capsid assembly (right panel).See also FIG. 8.

FIGS. 3A-E are a series of gels, charts, and schematics representingexperimental data demonstrating that the requirement for AAP exhibitsbranch specificity in the context of a putative AAV phylogeny. Panel Ais a graph showing that AAPstop60s were generated for the nine putativeancestral AAVs. Vector produced from WT or AAPstop60 rep-cap constructswas titrated by qPCR to quantify DNase resistant particles. AAPstop60titers are reported as a percentage of each serotype's WT titer andrepresent the mean of at least 3 independent experiments ±SEM. Bar colorcorresponds to heatmap color on y-axis and is used also in (Panel D) and(Panel E). † AAPstop60 titer below background (no cap gene control) forat least one trial. See Table 2 for statistics. * Anc126 produces atconsistently low titers (below 1e9 GC/mL) for WT and AAPstop60. Panel Bis a graph showing that AAPstop20s were generated for AAV4 and all AAVvariants with AAPstop60 titers ≥10% by introducing early stop codons at˜20 aa into the AAP ORF. Vector was produced and titrated as in Panel A,adding the AAPstop20 condition (light grey bars) and AAPstop20 plus aCMV-driven construct expressing a homologous AAP (dark grey bars) (meanof two experiments). † Titer below background (no cap gene control) forat least one trial. ‡ Rescue performed with AAP2. Panel C are gels ofHEK 293 cells transfected with helper and rep-cap plasmids as denotedabove lane: (wt) WT AAP; (s60) AAPstop60; (s20) AAPstop20; (r) AAPstop20plus CMV-driven homologous AAP. Whole cell lysates were harvested after36 hours and VP levels interrogated by Western Blot. Tubulin was used asa loading control. Panel D is the categorization of AAP phenotypes.Boxes below each serotype indicate AAPstop60 percentage of WT titer.Black boxes indicate AAP-independent (AAPstop20 titer is >>1%).Serotypes with AAPstop60 titers >10% (green) indicate assembly inabsence of C-terminal two-thirds of AAP (AAPC-independent). Panel E is areconstructed AAV phylogeny, branches colored as in Panel D. Greynumbers on branches indicate number of divergent amino acids between thetwo serotypes flanking the branch segment. See also FIGS. 8, 9, 10, and12.

FIGS. 4A-F are a series of schematics, graphs, and gels experimentaldata showing the characterization of 82DI, an AAPC-independentgain-of-function mutant. Panel A are schematics of 82DI generated byintroducing Branch I residue identities into an Anc82 rep-cap plasmid bysite directed mutagenesis. Panel B is a graph of vector produced fromAnc82, 82DI, and their AAPstop60s was titrated by quantifying DNaseresistant particles (DRP), and is reported as a percentage of Anc82 WTtiter. Graph represents the mean of four independent experiments, ±SEM.See Table 2 for statistics. Panel C is a graph, where each site in 82DIand 82DIAAPstop60 was reverted to its Anc82 identity individually bysite directed mutagenesis. Vector titers quantifying DRPs are reportedas a percentage of 82DI WT titer and represent the mean of 2 trials.Panel D is a photograph of a gel of HEK 293 cells transfected withhelper and rep-cap plasmids as denoted above lane: (WT) WT AAP; (s60)AAPstop60; (s20) AAPstop20; (r) AAPstop20 plus CMV-driven AAP2. Wholecell lysates were harvested after 36 hours, and VP levels interrogatedby Western Blot. Tubulin was used as a loading control. Panel E isnormalized SYPRO® Orange fluorescence signals obtained for Anc82 and82DI. Panel F are photographs of GFP fluorescence detected in murinelivers 30 d after systemic injection with 1×10¹¹ vg/mouse of Anc82,82DI, or AAV8. Each image is representative of an individual animal. Seealso FIGS. 12 and 13.

FIGS. 5A-F are a series of molecular-level and atomic-level schematicsdemonstrating that sites of interest map to the trimer interface,suggesting stronger inter-monomeric interactions in AAPC-independentserotypes. Panel A is a summary of the ten sites (twelve residues)identified by Branch D/Branch I multiple sequence alignment, numberedfrom VP1 start codon. Branch D residues include Anc80, Anc81, Anc82,Anc83, Anc84, AAV8, and rh10; Branch I residues include Anc110, rh8, andAAV9. Variations in identity for AAV8 and AAV9 are indicated inparentheses and are exclusive to these members of their respectivebranches. Panel B is a side view of an AAV9 trimer, showing planes ofview in Panel C. Each monomer is represented as one color, and each siteof interest in a darker shade of that color. Numbered arrows indicateeach site within the red monomer. Panels D-F are atomic-level views ofselect sites in AAV8 and AAV9 trimers.

FIGS. 6A-C are a series of schematics and gels of experimental datademonstrating that AAP promotes VP-VP interactions. Panel A is aschematic of expression constructs for AAP2 and VP1 and VP3 of AAV2,AAV3, Anc82, and 82DI. In CMV-HA-VP1, the VP2 and VP3 start codons weremodified to silence their expression, and the AAPstop60 mutation (redrectangle) was included. Panel B are photographs of HEK293 cellstransfected with CMV-HA-VP1 and CMV-VP3 of serotype indicated above eachlane, +/−CMV-AAP2, and lysates harvested after 48 h. Immunoprecipitationwas performed using anti-HA antibody; VPs detected by Western Blot usingthe B1 antibody. Panel C are photographs of lysates from CMV-HA-VP1,CMV-VP3, +/−CMV-AAP2 (all AAV2 proteins) transfected HEK293 cellstreated with DMSO, 5 mM disuccinimidyl glutarate (DSG), or 5 mMdisuccinimidyl suberate (DSS) as indicated above columns. VPs weredetected by Western Blot with B1 antibody. Approximate molecular weightsare shown to the right of each row. See also FIG. 13.

FIG. 7 is a schematic model for early steps of capsid assembly acrossthe AAP phenotypes. Whether a serotype is AAP-dependent,AAP-independent, or AAPC-independent, nucleating capsid assembly islikely dependent on both the stability and oligomerization of VPproteins. The findings herein demonstrate AAP is active in bothfunctions. Whether or not these functions are separate is unclear, andindicated by question marks in the model.

FIGS. 8A-B are a series of representations of gels showing AAV8 and AAV3VP degradation. Related to FIGS. 2A-E and FIGS. 3A-E. Panel A arephotographs of HEK 293 cells transfected with helper and AAV8 wt orAAPstop60 rep-cap plasmids as indicated. At 24 h, AAPstop60 transfectedcells were treated with 50 μM cycloheximide (CHX) and lysates harvestedat progressive time points. VP levels were interrogated by Western Blotwith B1 antibody, and p62 was blotted for as a positive control for CHXeffectiveness. The exposure shown for AAV8AAPstop60 transfected cells isa long exposure with a higher sensitivity detection reagent, todemonstrate that AAV8 VPs could not be detected in the absence of AAP.Panel B are photographs of HEK 293 cells transfected with helper andAAV3 wt or AAPstop20 rep-cap plasmids as indicated. At 24 h, AAPstop20transfected cells were treated with concentrations of Bortezomib,MLN7243, or Bafilomycin as indicated above lanes, and incubated anadditional 8 h before whole cell lysates were harvested. VP levels wereinterrogated by Western Blot with B1 antibody (top). Blot was strippedand reprobed for Ubiquitin (bottom). Actin was used as a loading control(middle).

FIG. 9 is a schematic showing the conservation of AAP across 21serotypes. Related to FIGS. 1A-D, 2A-F, 3, 6, and 7. Multiple proteinsequence alignment (ClustalW) for AAPs of all 21 serotypes examined inthis study. Ancestral sequence reconstruction to generate the Anc-AAVs(and by extension, Anc-AAPs) is detailed elsewhere (Zinn et al., 2015,Cell Rep., 12:1056-68). In brief, VP coding sequence for AncAAVs wasdetermined first on a protein level, and then reverse translated to DNAfor subsequent synthesis using a codon table from the most similarextant AAV sequence available. The conserved core (black bar) identifiedpreviously (Naumer et al., 2012, J. Virol., 86:13038-48) retains highconservation across the Anc-AAPs, and may confer the chaperone functionsuggested by the data for AAPN (purple bar) in the present work. Thework described herein additionally points to a scaffolding function forAAP that may be largely contained in the C-terminal two-thirds of AAP(AAPC, grey bar).

FIGS. 10A-C are a series of schematics and graphs of experimental datademonstrating that AAV3 AAPN does not rescue AAP-dependent viralproduction. Related to FIGS. 3A-E. Panel A are constructs expressingonly AAP generated by adding early stop codons in the VP1, VP2, and VP3ORFs of an AAV3 genome, and the AAPstop60 mutation was included togenerate a construct expressing only AAPN. The VP3 early stop codon is asilent mutation in the AAP ORF. Panel B is a graph from constructs in(A) used to trans-complement AAPstop20 viral production in AAV2 and AAV3(green and red bars) and viral titers reported as a percentage of theirWT titer. Graph represents average of two trials. † Titer belowbackground (no cap gene control) in at least one trial. Panel C showsthe individual data for both trials.

FIGS. 11A-D are a series of representations of microscope images andgraphs that show experimental data demonstrating that AAPstop60 virus isindistinguishable from wt AAP virus. Related to FIGS. 1A-E. Panel A arephotographs of AAV3 and AAV3AAPstop60.CMV.EGFP.T2A.Luciferase vectorstained with uranyl acetate and imaged by TEM. Panels B and C areresults from HEK293 cells incubated with hAd5 (MOI=20) overnight, thenAAV3, AAV3AAPstop60, AAV9 or AAV9AAPstop60.CMV.EGFP.T2A.Luciferase wasadded at GC/well indicated on x-axis in Panel C. GFP fluorescence wasimaged at 48 h (Panel B; images represent the highest titer for eachvector). Luciferase activity was quantified at 48 h (Panel C). Panel Dis normalized SYPRO® Orange fluorescence signals obtained for AAV3 andAAV9 WT and AAPstop60 vectors.

FIGS. 12A-B are a series of graphs that show experimental data of Anc82vs Anc82DI in vitro and in vivo as follows. Related to FIGS. 4A-F. PanelA is a graph of HEK293 cells incubated with hAd5 (MOI=20) overnight,then Anc82 or Anc82DI.CMV.EGFP.T2A.Luciferase vector added at 1×10⁹ or1×10⁸ GC/well as indicated. Luciferase activity was measured after 48 h.Panel B is a graph of mice injected systemically with 1×10¹¹ vg/mouse ofAnc82, 82DI, or AAV8.CB7.CI.EGFP.FF2A.hA1AT.RBG. Human α-1 antitrypsin(hA1AT) levels were measured by ELISA in serum sampled on time pointsindicated on x axis.

FIGS. 13A-B are a pair of bar graphs of experimental data showing thatthe IP fraction does not contain fully assembled capsids. Related toFIG. 6. Rep, helper, and ITR-CMV-EGFP-T2A-Luc_ITR reporter genomeplasmids were transfected with the AAV2 protein expression constructsindicated on x-axis. Fully assembled vectors in the input, IP, andsupernatant fractions were quantified by (Panel A) qPCR on DNaseresistant genomes or (Panel B) A20 capsid ELISA. Graphs arerepresentative of two independent experiments. † At least onemeasurement falls below the limit of detection.

DETAILED DESCRIPTION

Gene transfer, either for experimental or therapeutic purposes, reliesupon a vector or vector system to shuttle genetic information intotarget cells. The vector or vector system is considered the majordeterminant of efficiency, specificity, host response, pharmacology, andlongevity of the gene transfer reaction. Currently, the most efficientand effective way to accomplish gene transfer is through the use ofvectors or vector systems based on viruses that have been madereplication-defective. One of the most common viruses to be madereplication-defective and used in gene transfer is adeno-associatedvirus (AAV).

The AAV capsid is a non-enveloped, icosahedral 60-mer of three repeatingprotein monomer subunits called viral protein 1 (VP1), VP2, and VP3. Asingle transcript expressed from the AAV cap gene containing nested openreading frames (ORFs) is alternately spliced, resulting in threedistinct protein products that share C-terminal identity the length ofVP3. A 1:1:10 stoichiometry of VP1:VP2:VP3 in the assembled capsid isthought to be a consequence of the relative abundance of each protein,which is, in turn, regulated by splice product abundance and anon-canonical ACG translation start codon for VP2.

The Assembly-Activating Protein (AAP) is a non-structural proteinexpressed from a non-canonical CTG start codon of an overlapping readingframe embedded within the capsid (cap) gene of AAV. AAV serotypes havedifferent requirements for AAP, with some AAV serotypes exhibitingAAP-dependence (e.g., AAV8, rh10, Anc80, Anc81, Anc82, Anc83, and Anc84)and other AAV serotypes exhibiting AAP-independence (e.g., AAV9, rh8,and Anc110).

As used herein, an ancestral scaffold sequence refers to a non-naturallyoccurring sequence that is constructed using evolutionary probabilitiesand evolutionary modeling and is not known to have ever existed or topresently exit in nature. These scaffold sequences were leveraged hereinto interrogate AAP function and delineate structural determinants withinthe capsid relevant to the virus' requirement for AAP.

This disclosure provides methods of modifying the AAP-dependence of anAAV. For example, an AAV capsid sequence can be engineered to includethe motif identified herein, which reduces the AAP-dependence (or,conversely, increases the AAP-independence) during packaging of the AAV.This provides a number of benefits during manufacturing including,without limitation, the ability to reduce the number of componentsneeded for productive particle assembly in any AAV production system(e.g. mammalian, yeast, insect cell), the ability to optimize AAV capsidstructure with reduced constraints imposed by AAP, the potential of AAVcapsid self assembly from minimal components, and the reduction of AAPcontamination concerns in the final vector preparations.

Adeno-Associated Virus (AAV) Nucleic Acid and Polypeptide SequencesImparting Modified AAP-Dependency

A non-naturally occurring AAV capsid sequence, based originally on theAnc82 sequence (SEQ ID NO:1, encoded by SEQ ID NO:2, both shown below),which exhibits AAP-dependence during packaging, has been modified asdescribed herein to produce Anc82DI (SEQ ID NO:3, encoded by SEQ IDNO:4, both shown below). Anc82DI exhibits AAP-independence duringpackaging, but appears to retain functionality as a potent gene transfervector. The sequence motif that imparts AAP-independence onAAP-dependent sequences is provided in Table 1 above.

Anc82 protein (SEQ ID NO: 1)MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQREPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTAGTQTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSTTTNQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGNDNVDYSNVMITSEEEIKTTNPVATEEYGVVATNLQSANTAPQTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL Anc82 DNA (SEQ ID NO: 2)ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGGCCGGTAGAGCAGTCACCACAGCGTGAGCCCGACTCCTCCACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCCAAAAAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGATCTAATACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGTGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTCGGGAGGCAGCACCAACGACAACACCTACTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAGGTCAAAGAGGTCACGACGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCGTCCAGGTGTTTACGGACTCGGAATACCAGCTGCCGTACGTCCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGCTACCTGACTCTCAACAACGGTAGTCAGGCCGTGGGACGTTCCTCCTTCTACTGCCTGGAGTACTTCCCCTCTCAGATGCTGAGAACGGGCAACAACTTTCAATTCAGCTACACTTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGTTTGGACAGGCTGATGAATCCTCTCATCGACCAGTACCTGTACTACCTGTCAAGAACCCAGACTACGGGAGGCACAGCGGGAACCCAGACGTTGCAGTTTTCTCAGGCCGGGCCTAGCAGCATGGCGAATCAGGCCAAAAACTGGCTGCCTGGACCCTGCTACAGACAGCAGCGCGTCTCCACGACAACGAATCAAAACAACAACAGCAACTTTGCCTGGACTGGTGCCACCAAGTATCATCTGAACGGCAGAGACTCTCTGGTGAATCCGGGCGTCGCCATGGCAACCCACAAGGACGACGAGGACCGCTTCTTCCCATCCAGCGGCGTCCTCATATTTGGCAAGCAGGGAGCTGGAAATGACAACGTGGACTATAGCAACGTGATGATAACCAGCGAGGAAGAAATCAAGACCACCAACCCCGTGGCCACAGAAGAGTATGGCGTGGTGGCTACTAACCTACAGTCGGCAAACACCGCTCCTCAAACGGGGACCGTCAACAGCCAGGGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCTATTTGGGCCAAGATTCCTCACACAGATGGCAACTTTCACCCGTCTCCTTTAATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTCATCAAAAACACTCCTGTTCCTGCGGATCCTCCAACAACGTTCAACCAGGCCAAGCTGAATTCTTTCATCACGCAGTACAGCACCGGACAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAGCGCTGGAACCCAGAGATTCAGTATACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTTAATACTGAGGGTGTTTACTCTGAGCCTCGCCCCATTGGCACTCGTTACCTCACCCGTAATCTGTAAAnc82DI protein (SEQ ID NO: 3):MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQREPDSSTGIGKSGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTAGTQTLQFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGATKYHLNGRDSLMNPGVAMASHKDDEDRFFPSSGVLIFGKQGAGNDNVDYSNVMITSEEEIKTTNPVATEEYGVVATNHQSANTQAQTGTVQNQGILPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL Anc82DI DNA (SEQ ID NO: 4):ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGGCCGGTAGAGCAGTCACCACAGCGTGAGCCCGACTCCTCCACGGGCATCGGCAAGAGCGGCCAGCAGCCCGCCAAAAAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGATCTAATACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGTGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTAGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTCGGGAGGCAGCACCAACGACAACACCTACTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAGGTCAAAGAGGTCACGACGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCGTCCAGGTGTTTACGGACTCGGAATACCAGCTGCCGTACGTCCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGCTACCTGACTCTCAACAACGGTAGTCAGGCCGTGGGACGTTCCTCCTTCTACTGCCTGGAGTACTTCCCCTCTCAGATGCTGAGAACGGGCAACAACTTTCAATTCAGCTACACTTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGTTTGGACAGGCTGATGAATCCTCTCATCGACCAGTACCTGTACTACCTGTCAAGAACCCAGACTACGGGAGGCACAGCGGGAACCCAGACGTTGCAGTTTTCTCAGGCCGGGCCTAGCAGCATGGCGAATCAGGCCAGAAACTGGGTGCCTGGACCCTGCTACAGACAGCAGCGCGTCTCCACGACAACGAATCAAAACAACAACAGCAACTTTGCCTGGACTGGTGCCACCAAGTATCATCTGAACGGCAGAGACTCTCTGATGAATCCGGGCGTCGCCATGGCAAGCCACAAGGACGACGAGGACCGCTTCTTCCCATCCAGCGGCGTCCTCATATTTGGCAAGCAGGGAGCTGGAAATGACAACGTGGACTATAGCAACGTGATGATAACCAGCGAGGAAGAAATCAAGACCACCAACCCCGTGGCCACAGAAGAGTATGGCGTGGTGGCTACTAACCACCAGTCGGCAAACACCCAGGCTCAAACGGGGACCGTCCAAAACCAGGGAATCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCTATTTGGGCCAAGATTCCTCACACAGATGGCAACTTTCACCCGTCTCCTTTAATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTCATCAAAAACACTCCTGTTCCTGCGGATCCTCCAACAACGTTCAACCAGGCCAAGCTGAATTCTTTCATCACGCAGTACAGCACCGGACAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAGCGCTGGAACCCAGAGATTCAGTATACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTTAATACTGAGGGTGTTTACTCTGAGCCTCGCCCCATTGGCACTCGTTACCTCACCCGTAATCTGTAA

In addition to the polypeptides having the amino acid sequences shown inSEQ ID NOs: 1 and 3, polypeptides are provided that have at least 95%sequence identity (e.g., at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity) to the 55 polypeptides having theamino acid sequences shown in SEQ ID NOs: 1 and 3. Similarly, nucleicacid molecules are provided that have at least 95% sequence identity(e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity) to the nucleic acid molecules shown in SEQ ID NOs: 2and 4.

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of nucleotides or amino acid residuesbetween the two sequences is determined. The number of identical matchesis divided by the length of the aligned region (i.e., the number ofaligned nucleotides or amino acid residues) and multiplied by 100 toarrive at a percent sequence identity value. It will be appreciated thatthe length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. It alsowill be appreciated that a single sequence can align with more than oneother sequence and hence, can have different percent sequence identityvalues over each aligned region.

The alignment of two or more sequences to determine percent sequenceidentity can be performed using the algorithm described by Altschul etal. (1997, Nucleic Acids Res., 25:3389 3402) as incorporated into BLAST(basic local alignment search tool) programs, available atncbi.nlm.nih.gov on the World Wide Web. BLAST searches can be performedto determine percent sequence identity between a sequence (nucleic acidor amino acid) and any other sequence or portion thereof aligned usingthe Altschul et al. algorithm. BLASTN is the program used to align andcompare the identity between nucleic acid sequences, while BLASTP is theprogram used to align and compare the identity between amino acidsequences. When utilizing BLAST programs to calculate the percentidentity between a sequence and another sequence, the default parametersof the respective programs generally are used.

This disclosure also provides vectors containing nucleic acid moleculesthat encode polypeptides. Vectors, including expression vectors, arecommercially available or can be produced by recombinant technology. Avector containing a nucleic acid molecule can have one or more elementsfor expression operably linked to such a nucleic acid molecule, andfurther can include sequences such as those encoding a selectable marker(e.g., an antibiotic resistance gene), and/or those that can be used inpurification of a polypeptide (e.g., 6×His tag). Elements for expressioninclude nucleic acid sequences that direct and regulate expression ofnucleic acid coding sequences. One example of an expression element is apromoter sequence. Expression elements also can include one or more ofintrons, enhancer sequences, response elements, or inducible elementsthat modulate expression of a nucleic acid molecule. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin andvectors can contain a combination of expression elements from differentorigins. As used herein, operably linked means that elements forexpression are positioned in a vector relative to a coding sequence insuch a way as to direct or regulate expression of the coding sequence.

A nucleic acid molecule, e.g., a nucleic acid molecule in a vector(e.g., an expression vector, such as a viral vector) can be introducedinto a host cell. The term “host cell” refers not only to the particularcell(s) into which the nucleic acid molecule has been introduced, butalso to the progeny or potential progeny of such a cell. Many suitablehost cells are known to those skilled in the art; host cells can beprokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeastcells, insect cells, plant cells, mammalian cells). Representative hostcells can include, without limitation, A549, WEHI, 3T3, 10T1/2, BHK,MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293 cells,Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. Methods for introducing nucleic acid moleculesinto host cells are well known in the art and include, withoutlimitation, calcium phosphate precipitation, electroporation, heatshock, lipofection, microinjection, and viral-mediated nucleic acidtransfer (e.g., transduction).

With respect to polypeptides, “purified” refers to a polypeptide (i.e.,a peptide or a polypeptide) that has been separated or purified fromcellular components that naturally accompany it. Typically, thepolypeptide is considered “purified” when it is at least 70% (e.g., atleast 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from thepolypeptides and naturally occurring molecules with which it isnaturally associated. Since a polypeptide that is chemically synthesizedis, by nature, separated from the components that naturally accompanyit, a synthetic polypeptide is considered “purified,” but further can beremoved from the components used to synthesize the polypeptide (e.g.,amino acid residues). With respect to nucleic acid molecules, “isolated”refers to a nucleic acid molecule that is separated from other nucleicacid molecules that are usually associated with it in the genome. Inaddition, an isolated nucleic acid molecule can include an engineerednucleic acid molecule such as a recombinant or a synthetic nucleic acidmolecule.

Polypeptides can be obtained (e.g., purified) from natural sources(e.g., a biological sample) by known methods such as DEAE ion exchange,gel filtration, and/or hydroxyapatite chromatography. A purifiedpolypeptide also can be obtained, for example, by expressing a nucleicacid molecule in an expression vector or by chemical synthesis. Theextent of purity of a polypeptide can be measured using any appropriatemethod, e.g., column chromatography, polyacrylamide gel electrophoresis,or HPLC analysis. Similarly, nucleic acid molecules can be obtained(e.g., isolated) using routine methods such as, without limitation,recombinant nucleic acid technology (e.g., restriction enzyme digestionand ligation) or the polymerase chain reaction (PCR; see, for example,PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., ColdSpring Harbor Laboratory Press, 1995). In addition, isolated nucleicacid molecules can be chemically synthesized.

Methods of Making Virus Particles Having Modified AAP-Dependence

After the desired sequence of a virus or portion thereof has beendetermined (e.g., having a modified AAP-dependency), the actual nucleicacid molecule and/or polypeptide(s) can be generated, e.g., synthesized.Methods of generating an artificial nucleic acid molecule or polypeptidebased on a sequence obtained, for example, in silico, are known in theart and include, for example, chemical synthesis or recombinant cloning.Additional methods for generating nucleic acid molecules or polypeptidesare known in the art and are discussed in more detail below.

Once a polypeptide has been produced, or once a nucleic acid moleculehas been generated and expressed to produce a polypeptide, thepolypeptide can be assembled into a virus particle using, for example, apackaging host cell. The components of a virus particle (e.g., repsequences, cap sequences, inverted terminal repeat (ITR) sequences) canbe introduced, transiently or stably, into a packaging host cell usingone or more vectors as described herein.

Virus particles can be purified using routine methods. As used herein,“purified” virus particles refer to virus particles that are removedfrom components in the mixture in which they were made such as, but notlimited to, viral components (e.g., rep sequences, cap sequences),packaging host cells, and partially- or incompletely-assembled virusparticles.

Once assembled, the virus particles can be screened for, e.g., theability to replicate; gene transfer properties; receptor bindingability; and/or seroprevalence in a population (e.g., a humanpopulation). Determining whether a virus particle can replicate isroutine in the art and typically includes infecting a host cell with anamount of virus particles and determining if the virus particlesincrease in number over time. Determining whether a virus particle iscapable of performing gene transfer also is routine in the art andtypically includes infecting host cells with virus particles containinga transgene (e.g., a detectable transgene such as a reporter gene,discussed in more detail below). Following infection and clearance ofthe virus, the host cells can be evaluated for the presence or absenceof the transgene. Determining whether a virus particle binds to itsreceptor is routine in the art, and such methods can be performed invitro or in vivo.

Determining the seroprevalence of a virus particle is routinelyperformed in the art and typically includes using an immunoassay todetermine the prevalence of one or more antibodies in samples (e.g.,blood samples) from a particular population of individuals.Seroprevalence is understood in the art to refer to the proportion ofsubjects in a population that is seropositive (i.e., has been exposed toa particular pathogen or immunogen), and is calculated as the number ofsubjects in a population who produce an antibody against a particularpathogen or immunogen divided by the total number of individuals in thepopulation examined. Immunoassays are well known in the art and include,without limitation, an immunodot, Western blot, enzyme immunoassays(EIA), enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay(RIA). Simply by way of example, see Xu et al. (2007, Am. J. Obstet.Gynecol., 196:43.e1-6); Paul et al. (1994, J. Infect. Dis., 169:801-6);Sauerbrei et al. (2011, Eurosurv., 16(44):3); Boutin et al. (2010, Hum.Gene Ther., 21:704-12); Calcedo et al. (2009, J. Infect. Dis.,199:381-90); and Sakhria et al. (2013, PLoS Negl. Trop. Dis., 7:e2429),each of which determined seroprevalence for a particular antibody in agiven population.

As described herein, virus particles can be neutralized by a person's,e.g., patient's, immune system. Several methods to determine the extentof neutralizing antibodies in a serum sample are available. For example,a neutralizing antibody assay measures the titer at which anexperimental sample contains an antibody concentration that neutralizesinfection by 50% or more as compared to a control sample withoutantibody. See, also, Fisher et al. (1997, Nature Med., 3:306-12) andManning et al. (1998, Human Gene Ther., 9:477-85).

Methods of Using Viruses or Portions Thereof Having ModifiedAAP-Dependence

A virus or portion thereof that has a modified AAP-dependence asdescribed herein can be used in a number of research and/or therapeuticapplications. For example, a virus or portion thereof that has amodified AAP-dependence as described herein can be used in human oranimal medicine for gene therapy (e.g., in a vector or vector system forgene transfer) or for vaccination (e.g., for antigen presentation). Morespecifically, a virus or portion thereof that has a modifiedAAP-dependence as described herein can be used for gene addition, geneaugmentation, genetic delivery of a polypeptide therapeutic, geneticvaccination, gene silencing, genome editing, gene therapy, RNAidelivery, cDNA delivery, mRNA delivery, miRNA delivery, miRNA sponging,genetic immunization, optogenetic gene therapy, transgenesis, DNAvaccination, or DNA immunization.

A host cell can be transduced or infected with a virus or portionthereof having a modified AAP-dependence in vitro (e.g., growing inculture) or in vivo (e.g., in a subject). Host cells that can betransduced or infected with a virus or portion thereof having a modifiedAAP-dependence in vitro are described herein; host cells that can betransduced or infected with an ancestral virus or portion thereof invivo include, without limitation, brain, liver, muscle, lung, eye (e.g.,retina, retinal pigment epithelium), kidney, heart, gonads (e.g.,testes, uterus, ovaries), skin, nasal passages, digestive system,pancreas, islet cells, neurons, lymphocytes, ear (e.g., inner ear), hairfollicles, and/or glands (e.g., thyroid).

A virus or portion thereof having a modified AAP-dependence as describedherein can be modified to include a transgene (in cis or trans withother viral sequences). A transgene can be, for example, a reporter gene(e.g., beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase,thymidine kinase, green fluorescent polypeptide (GFP), chloramphenicolacetyltransferase (CAT), or luciferase, or fusion polypeptides thatinclude an antigen tag domain such as hemagglutinin or Myc) or atherapeutic gene (e.g., genes encoding hormones or receptors thereof,growth factors or receptors thereof, differentiation factors orreceptors thereof, immune system regulators (e.g., cytokines andinterleukins) or receptors thereof, enzymes, RNAs (e.g., inhibitory RNAsor catalytic RNAs), or target antigens (e.g., oncogenic antigens,autoimmune antigens)).

The particular transgene will depend, at least in part, on theparticular disease or deficiency being treated. Simply by way ofexample, gene transfer or gene therapy can be applied to the treatmentof hemophilia, retinitis pigmentosa, cystic fibrosis, leber congenitalamaurosis, lysosomal storage disorders, inborn errors of metabolism(e.g., inborn errors of amino acid metabolism including phenylketonuria,inborn errors of organic acid metabolism including propionic academia,inborn errors of fatty acid metabolism including medium-chain acyl-CoAdehydrogenase deficiency (MCAD)), cancer, achromatopsia, cone-roddystrophies, macular degenerations (e.g., age-related maculardegeneration), lipopolypeptide lipase deficiency, familialhypercholesterolemia, spinal muscular atrophy, Duchenne's musculardystrophy, Alzheimer's disease, Parkinson's disease, obesity,inflammatory bowel disorder, diabetes, congestive heart failure,hypercholesterolemia, hearing loss, coronary heart disease, familialrenal amyloidosis, Marfan's syndrome, fatal familial insomnia,Creutzfeldt-Jakob disease, sickle-cell disease, Huntington's disease,fronto-temporal lobar degeneration, Usher syndrome, lactose intolerance,lipid storage disorders (e.g., Niemann-Pick disease, type C), Battendisease, choroideremia, glycogen storage disease type II (Pompedisease), ataxia telangiectasia (Louis-Bar syndrome), congenitalhypothyroidism, severe combined immunodeficiency (SCID), and/oramyotrophic lateral sclerosis (ALS).

A transgene also can be, for example, an immunogen that is useful forimmunizing a subject (e.g., a human, an animal (e.g., a companionanimal, a farm animal, an endangered animal). For example, immunogenscan be obtained from an organism (e.g., a pathogenic organism) or animmunogenic portion or component thereof (e.g., a toxin polypeptide or aby-product thereof). By way of example, pathogenic organisms from whichimmunogenic polypeptides can be obtained include viruses (e.g.,picornavirus, enteroviruses, orthomyxovirus, reovirus, retrovirus),prokaryotes (e.g., Pneumococci, Staphylococci, Listeria, Pseudomonas),and eukaryotes (e.g., amebiasis, malaria, leishmaniasis, nematodes). Itwould be understood that the methods described herein and compositionsproduced by such methods are not to be limited by any particulartransgene.

A virus or portion thereof having a modified AAP-dependence, usuallysuspended in a physiologically compatible carrier, can be administeredto a subject (e.g., a human or non-human mammal). Suitable carriersinclude saline, which may be formulated with a variety of bufferingsolutions (e.g., phosphate buffered saline), lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, and water. The virus orportion thereof having a modified AAP-dependence is administered insufficient amounts to transduce or infect the cells and to providesufficient levels of gene transfer and expression to provide atherapeutic benefit without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to an organ such as, for example, theliver or lung, orally, intranasally, intratracheally, by inhalation,intravenously, intramuscularly, intraocularly, subcutaneously,intradermally, transmucosally, or by other routes of administration.Routes of administration can be combined, if desired.

The dose of the virus or portion thereof having a modifiedAAP-dependence that is administered to a subject will depend primarilyon factors such as the condition being treated, and the age, weight, andhealth of the subject. For example, a therapeutically effective dosageof a virus or portion thereof having a modified AAP-dependence that isto be administered to a human subject generally is in the range of fromabout 0.1 ml to about 10 ml of a solution containing concentrations offrom about 1×10¹ to 1×10¹² genome copies (GCs) of viruses (e.g., about1×10³ to 1×10⁹ GCs). Transduction and/or expression of a transgene canbe monitored at various time points following administration by DNA,RNA, or protein assays. In some instances, the levels of expression ofthe transgene can be monitored to determine the frequency and/or amountof dosage. Dosage regimens similar to those described for therapeuticpurposes also may be utilized for immunization.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1—Vectors and Sequences

Adeno-associated viral vectors were pseudotyped with either extant orancestral viral capsids. Extant capsids include AAV1 (Genbank [GB]AAD27757.1), AAV2 (GB AAC03780.1), AAV3 (GB U48704.1), AAV4 (GBU89790.1), AAV5 (GB AAD13756.1), AAV6 (GB AF028704.1), AAV7 (NC006260.1) Rh.10 (gb AA088201.1), AAV8 (GB AAN03857.1), AAV9 (GBAAS99264.1), and Rh32.33 (GB EU368926). Ancestral AAV capsids includeAnc80L65, Anc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, and Anc127(KT235804-KT235812). In this study, Anc83 has the following mutation inthe presumed AAP ORF: Q1L (83AAP-KI).

Example 2—Site Directed Mutagenesis

AAPstop60, AAPstop20, and 82DI single revertant mutations were generatedusing the QuikChange® II Site-Directed Mutagenesis Kit according to themanufacturer's instructions. To generate 82DI, the QuikChange® LightningMulti Site-Directed Mutagenesis Kit was used according to themanufacturer's instructions, in two phases: first, five sites weremutated on an Anc82 backbone, then the remaining five mutations wereintroduced into this quintuple mutant backbone.

Example 3—Crude Virus Preparations/Titration

Virus preparations to assay production in all serotypes and mutants wereprepared as follows: Polyethylenimine transfections of AAV cisITR-CMV-EGFP-T2A-Luc-ITR (2 μg), AAV trans rep-cap (2 μg), andadenovirus helper plasmid (4 μg) were performed on HEK 293 cells at 90%confluency in 6-well dishes. PEI Max (Polysciences)/DNA ratio wasmaintained at 1.375:1 (w/w) in serum-free media. Virus was harvestedafter 72 h by three freeze/thaw cycles followed by centrifugation at15000×g.

For DRP titers, crude preps were DNaseI treated, and resistant(packaged) vector genome copies were used to titrate preps by TaqManqPCR amplification (Applied Biosystems 7500, Life Technologies) withprimers and probes detecting CMV promoter regions of the transgenecassette.

Example 4—Thermostability Assay (AAV-ID)

Thermostability of purified vector was assayed by AAV-ID (Pacouret etal., 2017, Mol. Ther., 25:1375-86). Briefly, A 500 uL sample of SYPRO®Orange 50X was prepared using PBS²⁺ (21-030-CV, Corning Inc., Corning,N.Y.) as a solvent. 96-well plates were loaded with 45 uL samples,supplemented with 5 uL Sypro Orange 50X. PBS²⁺ and 0.25 mg/mL Lysozyme(L6876, SIGMA-ALDRICH, St. Louis, Mo., USA) solutions were used asnegative and positive controls, respectively. Plates were sealed andcentrifuged at 3000 rpm for 2 min, and subsequently loaded into a 7500Real-Time PCR System (ThermoFisher SCIENTIFIC). Samples were incubatedat 25° C. for 2 min prior to undergo a temperature gradient (25 to 99°C., ˜2° C./10 min, step and hold mode with 0.4° C. temperatureincrements), while monitoring the fluorescence of the SYPRO® Orange dyeusing the ROX filter cube available on both qPCR systems. Fluorescencesignals F were normalized between 0 and 100% and melting temperatureswere defined as the temperature for which the numerical derivative dF/dTreached its maximum.

Example 5—Enzyme-Linked Immunosorbent Assays

A20 capsid ELISAs were performed on crude virus preparations with thePROGEN AAV 2 Titration ELISA kit (ref #PRATV), according to themanufacturer's instructions.

hA1AT ELISAs were performed on 1:1250-1:10000 serial dilutions of mouseserum with the Cloud-Clone ELISA kit for α-1 antitrypsin (SEB697Hu, 96tests) according to the manufacturer's instructions.

Example 6—Animal Studies

C57BL/6 male mice (6-8 weeks) were purchased from Jackson Laboratories.All experimental procedures were performed in accordance with protocolsapproved by the Institutional Animal Care and Use Committee (IACUC) atSchepens Eye Research Institute.

Mice were anesthetized with Ketamine/Xylazine intraperitoneally. Eachanimal was injected retro-orbitally (100 μl) with 1.00E+11 VG/mouse ofthe following vectors: Anc82.CB7.CI.EGFP.FF2A.hA1AT.RBG andAnc82DI.CB7.CI.EGFP.FF2A.hA1AT.RBG. Blood was collected viasubmandibular bleeds using GoldenRod animal lancets (MEDIpoint, Inc.)prior injection, and 3, 7, 15 and 28 days post injection. Samples werecentrifuged at 8,000 rpm for 7.5 minutes and the serum was collected.

Animals were euthanized, and livers were collected and submerged in 4%paraformaldehyde solution (Electron Microscopy Sciences) for 30 minutes,then placed in 30% sucrose overnight. The next day the liver was mountedin Tissue-Tek O.C.T. Compound (Sakura Finetek) and flash frozen in coolisopentane.

Example 7—Tissue Histology

To visualize eGFP expression in liver, 15 μm sections were mounted withVECTASHIELD® Hard Set™ mounting medium with DAPI (H-1500) and imagedwith a Zeiss Axio Imager M2, at same gain and intensity across allsections.

Example 8—Molecular Representations

All molecular representations in this study were generated using PyMOLand Protein Data Bank files 2QA0 (AAV8) and 3UX1 (AAV9).

Example 9—Production and Purification of AAV3, AAV3s, AAV9 and AAV9s

Vectors were purified by affinity chromatography using either AVBSepharose HP (25-4112-11, GE Healthcare) (AAV3 and AAV3s) or POROSCaptureSelect AAV9 affinity resin (Thermo Fisher) per the manufacturerinstructions (AAV9 and AAV9s).

Large scale crude preps were treated with benzonase (250 U/mL, 1 h, 37°C.) before the centrifugation step (1 h, 10,000 rpm, 20° C.), thenfiltered using a 0.2 μm Nalgene Rapid-Flow filter. Vectors were purifiedby affinity chromatography using either HiTrap columns prepacked with 1mL AVB Sepharose HP (25-4112-11, GE Healthcare) (AAV3 and AAV3s) or a5×125 mm Econoline column (TAC05/125PE0-AB-3, essentialLife Solutions)packed with 1 mL POROS CaptureSelect AAV9 affinity resin (Thermo Fisher)per the manufacturer instructions (AAV9 and AAV9s). Columns weresanitized with 5 column volumes (CV) 0.1 M H₃PO4, 1 M NaCl, pH 2 (1mL/min) and equilibrated with 5 CV PBS (21-030-CV, Corning) (1 mE/min).Clarified lysates were injected at 1 mL/min.

Columns were further washed with 10 CV PBS (1 mL/min). Vector particleswere eluted in 3 mL 0.1 M NaOAc, 0.5 M NaCl, pH 2.5 (1 mL/min) andimmediately neutralized with 400 μL of 1 M Tris-HCl, pH 10. Samples werefurther buffer-exchanged in PBS and concentrated by Amicon filtration(UFC910024, EMD Millipore) per the manufacturer instructions. Samplepurity was assessed by SDS-PAGE, whereas DNAse I-resistant vectorgenomes were quantified by quantitative polymerase chain reaction (qPCR)using the TaqMan (Life Technologies) system with primers and probestargeting SV40 or eGFP.

Example 10—Statistical Methods

All data were analyzed using R prior to normalization for reporting inthe figures (unless otherwise indicated). P-values are reported in Table2, below. Viral titers were compared using a paired, one-tailedStudent's t test and RNA levels were compared using a paired, two-tailedStudent's t test.

TABLE 2 A FIG. 1C Serotype

m10

p-value

ND ND

ND FIG. 3A Serotype

p-value

ND ND ND ND ND FIG. 4B

Serotype

ND

ND

B FIG. 2C minor major

indicates data missing or illegible when filed

Table 2 shows the statistical analysis related to FIGS. 1A-D, 2A-E,3A-E, and 4A-F. See Table 3, below, for individual data of the titrationby ELISA and qPCR for these figures. All statistical analysis wasperformed in R, on data prior to normalization for reporting in the mainfigures indicated at left of table. Panel A compares WT and AAPstop60viral titers measured by qPCR after background subtraction (no cap genecontrol). P-values resulting from a paired, one-tailed t-test. ND=“notdetermined”; a t-test could not be performed on serotypes with one ormore trials within 3 standard deviations of measured background. Panel Bcompares AAPstop60 or rescue to WT levels of RNA (all normalized toGAPDH). P-values resulting from a paired, two-tailed t-test.

TABLE 3 AAV2 AAV3 WT AAPstop60 WT AAPstop60 Trial 1 GC/mL: qPCR 1.34E+102.64E+07 1.72E+10 2.89E+09 particles/mL: ELISA 2.48E+11 2.95E+086.14E+11 1.37E+11 % WT: qPCR 100 0.197 100 16.813 ELISA 100 0.119 10022.298 Trial 2 GC/mL: qPCR 8.25E+10 2.09E+08 1.76E+11 3.88E+10particles/mL: ELISA 6.78E+12 1.29E+09 2.23E+13 3.63E+12 % WT: qPCR 1000.253 100 22.045 ELISA 100 0.019 100 16.278 AAV2 AAV3 Average qPCR 1000.225 100 19.43  % wt: ELISA 100 0.069 100 19.29 

Example 11—Expression Constructs

AAP-HA: Complimentary oligonucleotides encoding the Hemagglutinin (HA)tag with BsiWI overhangs (5′ GTAC, 3′ CATG) were annealed in T4 LigaseBuffer ramping from 95° C. to 25° C. at 5°/min, PNK treated, and ligatedinto BsiWI digested and CIP treated AAV1 and AAV3 wt and AAPstop60rep-cap plasmids.

CMV-HA-VP1 and CMV-VP3: gBlocks® Gene Fragments (IDT) of bp #4-696 ofVP1 for AAV2, AAV3, Anc82, and 82DI were obtained, with the followingmodifications: an EcoRI site, start codon, and HA sequence added to 5′end, ACG to ACC mutation of VP2 start codon, and ATG to CTG mutation ofVP3 start codon. The gBlocks® include a BsrDI restriction site conservedin cap; gBlocks were digested with EcoRI and BsrDI. VP3 sequences werePCR amplified from the appropriate AAPstop60 rep-cap plasmids withprimers incorporating 5′ EcoRI and 3′ HindIII restriction sites, thendigested with either EcoRI and HindIII (for CMV-VP3) or BsrDI andHindIII (For CMV-HA-VP1). Fragments were ligated into pCDNA3.1(−) in theappropriate combinations. For CMV-AAP2, AAP was amplified from AAV2rep-cap plasmid and ligated into pCDNA3.1(−).

Example 12—Protein Lysate Preparation and Degradation/Turnover Studies

Transfections were performed as in Crude Virus Preparation. At 36 h,supernatant was aspirated and cells lysed on plate with 100 μL (FIGS.2A-E) or 150 μL (FIGS. 3A-E and 4A-F) lysis buffer (1% Triton X-100, 150mM NaCl, 50 mM Tris, pH8, plus cOmplete Mini™ protease inhibitor).Lysate was clarified by centrifugation at 15,000×g, diluted 1:50 inlysis buffer for actin or 1:100 for tubulin loading control blots, anddenatured in 4× NuPAGE® LDS sample buffer+0.5% OME at 90° C. 100 μg(FIGS. 2A-E) or 50 μg (FIGS. 3A-E and 4A-F) total protein (or dilutionthereof for loading control blot) per well were loaded andelectrophoresed on NuPAGE® 4-12% Bis-Tris gels.

For proteasome and lysosome inhibition experiments, media was removed 24h after transfection and replaced with media containing the appropriateconcentration of Bortezomib (Selleckchem PS-341), MLN7243 (ChemgoodC-1233), Bafilomycin (Enzo BML-CM110-0100), or DMSO (for wt and AAPstopuntreated samples) and incubated an additional 8 h. 25 μg total proteinwere loaded per well (diluted 1:10 for loading control). For proteinturnover experiments by blocking protein synthesis, media was removed 24h after transfection and replaced with media containing 50 μg/mL CHX(Sigma C7698) and lysates were harvested as described above at 1, 2, 4,6, and 8 h time points, and for the 0 h time point media was notreplaced but lysates were harvested. 10% FBS was maintained throughoutall transfections and drug incubations described here.

Example 13—Western/Dot Blotting

Electrophoresed proteins were transferred to PVDF membranes, incubatedwith primary antibody (B1, 1:250, ARP #03-65158; Actin, 1:20000, Abcam8227; Tubulin, 1:20000, Abcam 7291; HA, 1:5000, Abcam 9110; p62, 1:1000,Cell Signalling 5114) overnight, and detected with Anti-mouse (GEHealthcare LNXA931/AE) or Anti-rabbit (Sigma A0545) HRP conjugatedsecondary antibody and Thermo Super Signal® West Pico or Femto.

For dot blots, protein lysates were diluted 1:100 and 2 μL was spottedonto nitrocellulose membranes, allowed to dry, blocked in 5% milk, andincubated with ADK8 (ARP #03-651160) overnight.

Example 14—Immunoprecipitations

PEI transfections were performed with 10 μg each of CMV-HA-VP1, CMV-VP3,and CMV-AAP2 plasmid of the appropriate serotype (CMV-AAP2 added onlywhere indicated) on 10 cm dishes of HEK 293 cells at ˜80% confluency.PEI Max (Polysciences)/DNA ratio was maintained at 1.375:1 (w/w) inserum-free media. At 24 hours post transfection, cells were pelleted andresuspended in 1 mL lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mMTris, pH8, plus cOmplete Mini™ protease inhibitor). Immunoprecipitationwas performed with rb Anti-HA antibody (Abcam 9110) and Pierce ProteinA/G Plus Agarose beads. Precipitated proteins were eluted in 4× NuPAGE®LDS sample buffer+0.5% OME at 90° C. for 10 minutes. 10 μL (IP) or 30 μL(input) were loaded and electrophoresed on NuPAGE® 4-12% Bis-Tris gelsand detected in Western Blotting above.

For detection of full virions (FIG. 6C), ITR-CMV-EGFP-T2A-Luc-ITR andpRep plasmids were added to the above transfection with AAV2. To avoidheat-denaturation of AAV2 capsids, complexes were instead eluted with0.2 M glycine, pH 2.8, and eluates neutralized with equal volume Tris pH8.5. Purified AAV2 preps were treated in parallel with elution andneutralization buffers to ensure these conditions did not denature thecapsids. Control, IP, input, and supernatant fractions were DNaseItreated, and resistant (packaged) vector genome copies were quantifiedby TaqMan qPCR amplification (Applied Biosystems 7500, LifeTechnologies) with primers and probes detecting CMV promoter regions ofthe transgene cassette.

Example 15—Crosslinking

PEI transfections were performed with 2 μg each of CMV-HA-VP1, CMV-VP3,and CMV-AAP2 plasmid of the appropriate serotype (CMV-AAP2 added onlywhere indicated) on 6-well plates of HEK 293 cells at ˜80% confluency.PEI Max (Polysciences)/DNA ratio was maintained at 1.375:1 (w/w) inserum-free media. After 36 h, on-plate lysis was performed with M-PER™(Thermo) buffer supplemented with cOmplete™ mini protease inhibitor.Lysate was divided into three and treated with 5 mM final concentrationof disuccinimidyl glutarate (DSG, Thermo), disuccinimidyl suberate (DSS,Thermo), or an equal volume of DMSO as a mock treatment. Reactions wereincubated on ice for 1 h, mixing periodically, then quenched with 1 MTris. NuPAGE® LDS Sample Buffer+2-Mercaptoethanol were added and samplesboiled for 10 minutes, loaded onto SDS-PAGE gel, and interrogated byWestern Blot with B1 antibody.

Example 16—RNA Quantification

Transfections were performed as described in Crude VirusPreparations/Titration. RNA was harvested after 36 h with Qiagen RNeasyMini® kit according to the manufacturer's instructions. TURBO DNA-Free™kit (Invitrogen) was used according to manufacturer's instructions(Rigorous DNase treatment protocol) to eliminate contaminating DNA fromsamples. cDNA synthesis was performed with iScript™ kit (BioRad) using250 ng total RNA from each sample. Reactions were then diluted 10-fold,and 5 μL of diluted cDNA used in each qPCR reaction, prepared withPowerUp™ SYBR™ Green Master Mix (Applied Biosystems) and intron,intron-spanning, or GAPDH primers to detect unspliced, spliced, orhousekeeping gene products.

Example 17—Transmission Electron Microscopy

For negative staining on chromatography-purified AAV3 and AAV3stop,vector samples (5 μL or 50 μL drop) and a blank buffer control wereadsorbed onto 200 mesh carbon and formvar coated nickel grids, rinsed,and stained with 2% aqueous uranyl acetate for 30 seconds then absorbedoff on filter paper and air dried. All grids were imaged using a FEITecnai G2 Spirit transmission electron microscope (FEI, Hillsboro,Oreg.) at 100 kV accelerating voltage, interfaced with an AMT XR41digital CCD camera (Advanced Microscopy Techniques, Woburn, Mass.) fordigital TIFF file image acquisition. TEM imaging of AAV samples wereassessed and digital images captured at 2 k×2 k pixel, 16-bitresolution.

Example 18—In Vitro Transduction

The appropriate serotype and GC particles of AAV-CMV-EGFP-T2A-Luc wasadded to HEK-293 cells on a 96-well plate pre-infected with humanadenovirus 5 (hAd5) 24 h prior at a multiplicity of infection of 20particles/cell. The cells were imaged with an EVOS® FL Imaging System at24 and 48 h, after which D-luciferin containing buffer was added andluminescence was measured using Synergy H1 microplate reader (BioTek;Winooski, Vt.).

Example 19—Requirement for AAP Ranges Broadly Across all AAV Clades

To test whether AAP is required to assemble capsids from the fullcomplement of VP proteins (i.e., VP1, 2, and 3), AAP expression wasabolished from rep-cap trans plasmids by an early stop codon in the AAPreading frame, a silent mutation in VP (FIG. 1A, AAPstop60). AAPstop60swere generated for 12 serotypes, including at least one member of eachAAV clade, with the aim of a comprehensive assessment of AAP requirementacross mammalian AAV serotypes. Considering AAP's non-canonical CTGstart codon, AAPstop60 mutations were positioned such that they would besufficiently downstream of potential alternate start codons, yetupstream of regions shown to be essential for AAP2 function (Naumer etal., 2012, J. Virol., 86:13038-48). To verify loss of AAP protein, aHemagglutinin tag was inserted in the C-terminal region of the AAP ORFin two representative serotypes (FIG. 1A, AAP-HA). Whole cell lysatestransfected with these constructs were analyzed by Western Blot (FIG.1B), confirming that AAPstop60 results in loss of full-length AAP or anyshorter protein product translated from alternate starts. A double bandin the AAP-HA lane supports the likelihood of additional downstreamstart codons and corroborates the late placement of the stop codons(FIG. 1B).

Recombinant AAV vectors were produced from AAPstop60 and wildtype AAP(WT) plasmids, and titrated by qPCR quantifying DNase resistantparticles (DRP). AAPstop60 vector titers reveal that when all three VPproteins are present, AAP is not strictly required to assemble thevirion in several serotypes (FIG. 1C). Rather, AAP requirement rangesbroadly across serotypes, with AAPstop60 vectors producing as high as39% of WT titer for rh32.33, and as low as 0.035% WT titer for AAV8.This observation is in contrast with previous findings demonstratingAAP's absolute requirement for assembling VP3-only capsids, inparticular AAV9 and AAV1 (Sonntag et al., 2011, J. Virol., 85:12686-97).

The advantage of DRP titration is the ability to quantify virus of anycapsid serotype with the same vector genome absent of differential biasin measurement. However, DRP measures the amount of assembled particlesthat also underwent viral genome packaging, a process that occursdownstream of capsid assembly. Moreover, DRP does not assess fornon-packaged, empty AAV virions. To directly assay capsid assembly andrule out the possibility that serotypes with low AAPstop60 titers weredue to a packaging defect, an A20 capsid ELISA was performed, whichrecognizes a conformational epitope only present in assembled AAV2capsids. A20 cross-reacts with AAV3, allowing us to assay assemblydirectly for an AAV that requires AAP (AAV2) and one that accomplishesassembly in the AAPstop60 context (AAV3). A20 ELISA data for bothserotypes corroborated the DRP indirect measure of assembly (FIG. 1D).

Example 20—A Role for AAP in VP Protein Stability

To ensure that the observed range of AAP dependence for assembly was notdue to variation in VP translation efficiencies imposed by alternatecodon usage in the AAPstop60 mutants, VP protein levels produced by WTand AAPstop60 constructs were interrogated. The B1 monoclonal antibodydetects a conserved linear epitope on VP proteins in denaturingconditions across all AAVs tested in this study except AAV4 and rh32.33,allowing nearly all serotypes to be assayed. No appreciable differencein VP2 or VP3 protein levels, and a slight decrease in VP1 levels, wasobserved for AAPstop60s that produce 10% or higher of their respectiveWT titers (FIG. 2A), whereas AAPstop60s with titers below this thresholdshow a dramatic decrease in VP protein levels (FIG. 2B).

To examine whether the observed decreases in VP levels were due to apotential translational defect, AAP2 was co-expressed in trans withAAPstop60 (rescue; FIG. 2B). Appreciable restoration of VP protein wasobserved for all affected serotypes. Furthermore, no difference wasobserved between WT, AAPstop60, or rescue transcript levels (FIG. 2C),indicating that VP protein loss in the absence of AAP most likely occurspost- (or co-) translationally.

To interrogate degradation as the mechanism for instability, AAV8AAPstop60 transfected cells were treated with increasing concentrationsof the proteasome inhibitor Bortezomib, the E1 inhibitor MLN7243, or thevacuolar specific H+ ATPase inhibitor Bafilomycin (FIG. 2D). Thisallowed the examination of the earliest and latest steps of theUbiquitin-Proteasome Pathway, as well as late steps of lysosomaldegradation or autophagy by inhibiting the required acidification.Inhibiting lysosomal acidification resulted in a mild yet dose-dependentrescue of AAV8 VP3 protein. Proteasomal inhibition is accompanied by arobust rescue in AAV8 VP proteins in a dose dependent manner, but thiswas not concomitant with rescue of assembled capsids (FIG. 2E). E1inhibition provided an equally mild to moderate VP rescue independent ofdrug concentration. Collectively, these results suggest that instabilityof VP proteins in the absence of AAP can primarily be attributed toproteasomal degradation, and that this may in part beUbiquitin-independent. Lysosomal or autophagosomal degradation may alsodegrade a proportion of VP proteins.

In an attempt to examine the rate of AAV8 VP degradation, proteinsynthesis was blocked with Cycloheximide (CHX) and protein lysates wereharvested at progressive time points (FIG. 8A). As expected in AAPstop60lysates, VP protein levels were too low to detect even without CHXtreatment and despite long exposure times with highly sensitivedetection reagents. However, in the presence of AAP, VP protein levelsremain consistent over all time points of CHX treatment. This is likelybecause capsids are assembled rapidly in the presence of AAP, andbecause assembled VP proteins are not susceptible to degradation, a VPband persists.

Given the spectrum of AAP phenotypes observed across the major clades, 9putative evolutionary intermediates (AncAAVs) to the major AAV serotypesalso were tested in order to gain insight into what elements of VPstructure either impose the observed requirement for AAP or impart anability for some VPs to perform these functions independently (Zinn etal., 2015, Cell Rep., 12:1056-68). As with the natural serotypes, abroad range of requirement for AAP was observed for the AAPstop60AncAAVs (FIG. 3A). See Table 4 for individual AAPstop60, stop20, and AAPrescue vector titration data for FIGS. 3A-3E).

TABLE 4 % WT titers AAPstop60 AAPstop20 AAPstop20 + AAP Trial 1 Anc113126.757 -0.030 61.254 Rh32.33 52.415 96.577 114.124 AAV3 27.085 0.05061.983 Anc110 20.791 −0.037 34.955 AAV9 23.145 −0.013 28.922 AAV5 22.5126.297 83.410 AAV4 5.429 5.662 54.170 Trial 2 Anc113 253.371 0.359 46.539Rh32.33 39.059 89.731 105.907 AAV3 28.319 0.840 78.249 Anc110 23.7710.281 39.816 AAV9 8.769 −0.010 26.927 AAV5 14.108 6.912 79.285 AAV44.204 5.080 5.109 Average Anc113 190.064 0.165 53.897 Rh32.33 45.73793.154 110.016 AAV3 27.702 0.445 70.116 Anc110 22.281 0.122 37.386 AAV915.957 −0.012 27.924 AAV5 18.310 6.605 81.348 AAV4 4.816 5.371 29.639

Although the AAPstop60 early stop codon is placed upstream of domainsshown to be required for AAP2 function, it is downstream of a highlyconserved region (residues 52-57) in AAP (FIG. 9, conserved core). Thisdomain was shown by deletion analyses to be important for AAP2 function,but not sufficient to assemble VP3 only capsids without more C-terminalportions of AAP present (Naumer et al., 2012, J. Virol., 86:13038-48).Because AAPs in other serotypes have not yet been tested by deletionanalyses, and because algorithms that generated the AncAAVs were appliedonly to the VP ORF and may have unpredictable consequences on AAP (Zinnet al., 2015, Cell Rep., 12:1056-68), we wanted to examine whether apartially functional, N-terminal AAP (AAPN) was expressed from someAAPstop60 constructs, contributing to the observed varying requirementfor AAP across the 21 AAVs that were examined.

For the six AAVs whose AAPstop60 produces at least 10% of WT titer andfor AAV4, recently demonstrated to assemble VP3-only capsids without AAP(Earley et al., 2017, J. Virol., 91:e01980-16), further upstream stopconstructs (AAPstop20) were generated, placing the early stop codon atresidue ˜23 in the AAP ORF (silent mutations in VP). Of these, AAV5,rh32.33, and AAV4 AAPstop20 produce virus, while AAV3, AAV9, Anc 10, andAnc 13 do not (FIG. 3B). Although the B1 antibody does not detect AAV4and rh32.33, levels of VP protein produced from the remaining AAPstop20constructs mirror the titer (FIG. 3C).

Taken together with FIGS. 2A-E, these results demonstrate that stabilityis a serotype-specific property of VP proteins that fall into one ofthree categories: (i) independently stable, (ii) require only AAPN forstability, or (iii) requiring full-length AAP. These results clearlyillustrate a role for AAP in VP stability and provide an explanation forthe broad range of requirement for AAP. The discrepancy betweenAAPstop60 and WT titers, particularly for serotypes requiring only AAPN,points to additional shortcomings in some serotypes' VPs for which AAPcompensates, and potentially discrete functions contained primarily inAAPN versus the C-terminal two-thirds of AAP (AAPC).

Taking into consideration VP stability, AAPstop60 titers, and AAPstop20titers, for clarity, the AAP phenotypes were categorized as (i)AAP-independent, (ii) AAPC-independent, and (iii) AAP-dependent (FIG.3D). Additionally, it was shown that the AAPC-independent phenotype is aproperty of the VP proteins and not a result of a fully functional AAPNby demonstrating that AAPN of an AAPC-independent serotype cannot rescueviral production of an AAP-dependent serotype (FIG. 10).

To examine whether serotypes with different AAP phenotypes' VPs aresubject to the same mechanisms of degradation, degradation of AAV3proteins were blocked in the same manner as previously performed forAAV8 (FIG. 8B). Proteasome inhibition with Bortezomib provided adose-responsive, robust rescue as with AAV8, and E1 inhibition withMLN7243 rescued VP at the highest dose. Unlike AAV8, InhibitingLysosomal acidification with Bafilomycin robustly rescued AAV3 VP levelsin a dose dependent fashion, indicating that AAPC-independent serotypes(or at least AAV3) may be more susceptible to Lysosomal degradation orautophagy. These results could also suggest that AAPN somehow promotesthe proteasome as the primary means of degradation, whether by blockinglysosomal degradation or by other means, because AAPN is present in theAAV8 AAPstop60 lysates but is absent in the AAV3 AAPstop20 lysates.

Example 21—AAPC does not Impact Virion Morphology, Infectivity, orStability

While AAPN alone facilitates the production of appreciable quantities ofvirus for many serotypes, whether AAPN-assembled particles retain theproper morphology as well as infectivity functions was next addressed.TEM imaging of AAV3 WT and AAV3AAPstop60 vectors indicate identicalgross particle morphologies (FIG. 11A). To examine whether AAPC lossaffected infection capabilities, AAV9, AAV3, and their AAPstop60 vectorswere tested on HEK293 cells in culture (FIGS. 11B-C), demonstrating thatvirus assembled without AAPC retain infection capabilities.Additionally, the melting temperature of these particles were tested,and no appreciable difference was observed (FIG. 11D).

Example 22—Requirement for AAP Exhibits Branch Specificity in theContext of a Putative AAV Phylogeny

As a next step toward identifying VP structure responsible for assemblyfunctions, an overview of how AAP phenotypes diverge across the widegenetic range of AAV capsids tested was sought, aiming to identifyphenotypic differences across small genetic distances. AAP phenotypes ofthe 12 natural serotypes and the nine ancestral variants were correlatedto the reconstructed phylogeny (FIG. 3E). This revealed branch-specificAAP dependence profiles, with phylogenetic nodes illustrating cleardivergence in AAP phenotype. Among other apparent trends, Anc80, Anc81,Anc82, Anc83, and Anc84 comprise a fully dependent lineage thatterminates in AAV8 and rh.10, and will thus be referred to as Branch D(FIG. 3E, red arrow). At the Anc82 node, a phenotypic switch fromAAP-dependent to AAPC-independent is observed in its successor Anc 10.The serotypes that diverge from Anc 10, rh8 and AAV9, are alsoAAPC-independent; this branch was named Branch I (FIG. 3E, green arrow).

Example 22—Phenotype-to-Phylogeny Mapping Analysis Reveals a Set ofResidues Functioning in AAPC-Independent Assembly

The observation that AAP phenotypes have branch-specific trends withinthe phylogeny presented the opportunity for a facile method to identifyelements of VP structure critical for assembly functions. Given that VPsequence diverges by small increments along each of these branches, itwas hypothesized that a set of residues homologous only within themembers of their respective branch were likely to functionallycontribute to capsid assembly. To this end, a multiple sequencealignment was generated with the Branch D and Branch I members. Withinthe alignment, a total number of 149 positions varied, however, at onlytwelve positions, the residue is conserved within Branch I, with adifferent yet shared identity on Branch D. Of these twelve, eightindividual residues and two pairs of adjacent residues comprise 10 siteson VP. At some of these sites, residue identity diverges within Branch Imembers; however, they share a chemical property that contrasts withBranch D. For example, site 1 is a basic lysine in Branch D serotypes,compared to a threonine in Branch I for Anc 10 and rh8, and a serine inAAV9, both hydroxylic residues. The approach to identify a phenotypicswitch along a reconstructed phylogeny and then interrogate theconserved differences across the two diverging lineages for thephenotype of interest in order to map the structural determinant(s)responsible was named phenotype-to-phylogeny mapping.

Example 24—A Functional Motif Conferring AAPC-Independent Assembly andVP Protein Stability is Transferable to a Heterologous Capsid

To test whether the 10 sites constitute a motif that functions in capsidassembly, the Branch I identities were engrafted onto a member of BranchD and tested to determine whether the resulting hybrid gainsAAPC-independent assembly function. Anc82, the node from which Branch Idiverges, was chosen as the background for these mutations; as theclosest relative to the Branch I serotypes it is more likely to tolerateseveral targeted mutations and retain functionality than a more distantrelative. All ten sites in Anc82 were mutated to Branch I identities enmasse by site-directed mutagenesis, creating a variant named 82DI (FIG.4A). 82DIAAPstop60 gained AAPC-independent assembly function (FIG. 4B).To determine the minimal motif required to confer this phenotype, eachsite was individually reverted back to its Branch D identity. Allrevertants are AAP-dependent (FIG. 4C), corroborating the 10 sitesidentified using phenotype-to-phylogeny mapping not only constitute afunctional motif critical for capsid assembly, but also comprise aminimal motif required for AAPC-independent assembly in this subset ofserotypes.

Whether this DI motif affects VP protein stability was next assessed.Consistent with other AAP-dependent serotypes (FIG. 2B), Anc82 exhibitsa dramatic decrease in VP levels in the AAPstop60 condition, whereas82DIAAPstop60 does not (FIG. 4D; see Table 5 for individual 82DI singlerevertant vector titration data.

TABLE 5 % DI WT titer Trial 1 Trial 2 Average WT AAPstop WT AAPstop WTAAPstop site 82DI 100.000 12.047 100.000 16.941 100.00 14.494 1 S163K140.290 0.158 91.977 −0.008 116.134 0.075 2 S206A 128.656 −0.013 56.219−0.009 92.438 −0.011 3 R478K 154.220 −0.035 76.595 −0.008 115.408 −0.0224 V481L 95.214 0.033 56.655 −0.009 75.935 0.012 5 M520V 7.970 0.0170.382 −0.010 4.176 0.003 6 S528T 123.339 −0.064 83.723 −0.019 103.531−0.042 7 H586L 44.444 −0.035 18.062 −0.008 31.253 −0.021 8 QA592AP157.428 −0.024 122.904 −0.013 140.166 −0.019 9 QN599NS 120.802 0.02983.228 −0.019 102.015 0.005 10 I603A 110.992 0.019 90.107 −0.024 100.549−0.003

To properly categorize 82DI's AAP phenotype, AAPstop20 for Anc82 and DIwere generated, and loss of protein in 82DIstop20 was observed,indicating that 82DI is AAPC-independent (FIG. 4D).

To assess the broader impact of AAPC-independent assembly on the capsidas a whole, 82DI's biophysical properties and transduction capabilitieswas further characterized compared to its parental strain, Anc82. TheT_(m) of 82DI is 5° C. lower than Anc82 (FIG. 4E), a primary indicationof a biophysically distinct entity (Pacouret et al., 2017, Mol. Ther.,25:1375-86). Considering the marked changes in Anc82 vs. 82DI's T_(m)and AAP phenotypes, the infectivity of both variants was tested. 82DIretains infectivity and transduction may be increased moderatelycompared to Anc82 both in vitro (FIG. 12A) and in vivo (FIGS. 4F & 12B).

Example 25—Candidate Residues Contributing to AAP-Independent AssemblyLie at the VP Trimer Interface

To examine how this motif influences particle assembly, where theseresidues lie within the 3-dimensional fold of VP and within an assembledcapsid were mapped. Although crystal structures of AncAAVs are notavailable, the terminal Branch D (AAV8) and Branch I serotypes (AAV9)have been solved (DiMattia et al., 2012, J. Virol., 86:6947-58; Nam etal., 2007, J. Virol., 81:12260-71), and were used as surrogates to mapthe DI motif. Two of the 10 sites lie in the unstructured region of theVP N-terminus, but only site 1 is outside of VP3. Of the eight siteswithin the structured region of VP, seven of them map to the three-foldinterface of a VP trimer and contact a neighboring monomer (FIGS. 5A-C).

Comparing an AAV8 (AAP-dependent) trimer to an AAV9 (AAPC-independent)trimer at the atomic level, most of these sites exhibit compellingevidence for stronger inter-monomeric interactions within anAAPC-independent trimer than in the AAP-dependent trimer (FIGS. 5D-F).For example, a conserved glutamic acid forms a salt bridge with anadjacent monomer's histidine at site 7 in the AAV9 trimer that cannotform with the leucine at site 7 in AAV8 (FIG. 5D).

On AAV9, a hydrogen bond forms between a conserved asparagine and aglutamine at site 8 of an adjacent VP monomer. On AAV8, this bond isunable to materialize due to a Gln to Ala substitution (FIG. 5E). Site10 lies at the 3-fold axis, and beneath a conserved phenylalanine, thevaline residues in AAV9 create a much larger network of hydrophobicinteractions than the alanines in AAV8 (FIG. 5F). Moreover, the site 10interaction exists between all three monomers of the trimersimultaneously. These observations suggest that in AAPC-independentserotypes, this motif aids in trimer stabilization and possiblynucleates capsid assembly in the absence of a full length AAP.

Example 26—AAPC-Independent Capsomer Nucleation

Next, it was hypothesized that VPs of AAPC-independent AAVs are able toassociate into oligomers in the absence of AAPC, whereas AAP-dependentserotypes' VPs do not strongly associate unless a full-length AAP ispresent. To test this theory, VP-VP interaction of AAP-dependent andAAPC-independent AAVs were evaluated by co-immunoprecipitation of VP3with HA-tagged VP1 as bait (FIGS. 6A-B). The AAPC-independent VP1stested, AAV3 and 82DI, were able to co-precipitate VP3 in the absence ofa full-length AAP, despite low VP3 levels in the input. Conversely,neither AAP-dependent AAV2 nor Anc82 VP1 co-precipitated significant VP3despite appreciable input levels (FIG. 6B).

Addition of AAP2 allowed VP3 co-precipitation in AAP-dependentserotypes, and co-precipitated an unknown VP species between VP2 andVP3's predicted molecular weights in the AAPC-independent serotypes(marked with *, FIG. 6B). These may be VP2-like proteins translated froman alternate start codon, or VP1 N-terminal cleavage/degradationproducts stabilized by AAP. Despite the appreciable increase in Anc82and AAV3 VP3 input levels in the +AAP condition, these data support theabove hypothesis.

To begin examining whether AAP is promoting oligomerization into speciesof defined geometry such as trimers or pentamers, or whether theincrease in VP-VP interactions observed by co-IP are more randomizedassociations, crosslinking agents were added to transfected cell lysatesand VP species interrogated by Western Blot (FIG. 6C). In the presenceof AAP, a single supershifted VP band appears around 97 kDa when DSG (7angstrom crosslinking arm) was added, and a slightly larger supershifteddoublet appears when DSS (11 angstrom crosslinking arm) was added.Although it is difficult to determine the molecular weight ofcrosslinked species due to unpredictable migration patterns, thediscrete banding suggests that AAP promotes VP-VP interactions ofdefined geometry or number of monomers, but may also be indicative ofincreased association of VP with a host protein(s) involved in capsidassembly, an association promoted by AAP.

To further ensure that the VP oligomerization process was beinginterrogated separately from their assembly into full capsids, the IPexperiment was repeated with AAV2 VPs, adding rep, aap2, helper, andITR-flanked reporter genome plasmids in trans to allow quantification ofassembled DRPs (FIG. 13A). Appreciable quantities of genomes weredetected only in the input and supernatant fractions, but absent fromthe IP fraction. ELISA to quantify fully assembled capsids mirroredthese results (FIG. 13B), indicating only oligomerized VPs wereprecipitated. Taken together, these data support that in addition to arole for AAP in VP protein stability, AAP also promotes oligomerizationof VP proteins to nucleate assembly of the icosahedron, which couldpotentially increase the efficiency of the capsid assembly process.

Other Embodiments

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The disclosed methods, compositions, and other materials are disclosedas described herein, but it is understood that combinations, subsets,interactions, groups, etc. of these methods, compositions, and othermaterials are also disclosed. That is, while specific reference to eachvarious individual and collective combinations and permutations of thesecompositions and methods may not be explicitly disclosed, each isspecifically contemplated and described herein. For example, if aparticular composition of matter or a particular method is disclosed anddiscussed and a number of compositions or methods are discussed, eachand every combination and permutation of the compositions and themethods are specifically contemplated unless specifically indicated tothe contrary. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed.

What is claimed is:
 1. An adeno-associated virus (AAV) capsidpolypeptide comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 3.